Cancers 2021, 13, 4981. https://doi.org/10.3390/cancers13194981 www.mdpi.com/journal/cancers Article DEN‐Induced Rat Model Reproduces Key Features of Human Hepatocellular Carcinoma Keerthi Kurma 1,2 , Olivier Manches 2,3 , Florent Chuffart 1,2 , Nathalie Sturm 4 , Khaldoun Gharzeddine 1,2,5 , Jianhui Zhang 1,2 , Marion Mercey‐Ressejac 1,2,6 , Sophie Rousseaux 1,2 , Arnaud Millet 1,2,6 , Herve Lerat 1,2,7 , Patrice N. Marche 1,2 , Zuzana Macek Jilkova 1,2,6, * and Thomas Decaens 1,2,6, * 1 Université Grenoble Alpes, 38000 Grenoble, France; keerthi.kurma@chu‐montpellier.fr (K.K.); florent.chuffart@univ‐grenoble‐alpes.fr (F.C.); kgharzeddine@chu‐grenoble.fr (K.G.); zhangjh20@fudan.edu.cn (J.Z.); mressejac@chu‐grenoble.fr (M.M.‐R.); sophie.rousseaux@univ‐grenoble‐alpes.fr (S.R.); arnaud.millet@inserm.fr (A.M.); herve.lerat@univ‐grenoble‐alpes.fr (H.L.); patrice.marche@inserm.fr (P.N.M.); 2 Institute for Advanced Biosciences, Research Center Inserm U 1209/CNRS 5309, 38700 La Tronche, France; Olivier.Manches@efs.sante.fr 3 Etablissement Français du Sang, Rhone‐Alpes Auvergne, 38043 Grenoble, France 4 Service d’anatomo‐pathologie, Pôle de Biologie, CHU Grenoble Alpes, 38700 La Tronche, France; NSturm@chu‐grenoble.fr 5 Research Department, CHU Grenoble Alpes, 38700 La Tronche, France 6 Service d’hépato‐gastroentérologie, Pôle Digidune, CHU Grenoble Alpes, 38700 La Tronche, France 7 Unité mixte de service Université Grenoble Alpes hTAG, Inserm U046, CNRS UAR2019, 38700 La Tronche, France * Correspondence: ZMacekjilkova@chu‐grenoble.fr (Z.M.J.); tdecaens@chu‐grenoble.fr (T.D.) Simple Summary: Hepatocellular carcinoma is the most frequent form of primary liver cancer, characterized by increasing incidence and high mortality. Animal models of hepatocellular carci‐ noma are widely used to study the biology of cancer and to test potential therapies. Herein, we describe how the rat model of DEN‐induced hepatocellular carcinoma mimics the pathogenesis of hepatocellular carcinoma seen in humans, including liver damage, chronic inflammation, hepato‐ cytes proliferation, liver fibrosis and cirrhosis, disorganized vasculature, and modulations of the liver’s immune microenvironment. Our results should help the hepatocellular carcinoma field to better tailor the use of the DEN‐induced rat liver cancer model for testing specific experimental hypotheses or to perform preclinical testing. Abstract: Hepatocellular carcinoma (HCC) is the most common type of liver cancer. The majority of HCC cases are associated with liver fibrosis or cirrhosis developing from chronic liver injuries. The immune system of the liver contributes to the severity of tissue damage, the establishment of fibrosis and the disease’s progression towards HCC. Herein, we provide a detailed characterization of the DEN‐induced HCC rat model during fibrosis progression and HCC development with a spe‐ cial focus on the liver’s inflammatory microenvironment. Fischer 344 male rats were treated weekly for 14 weeks with intra‐peritoneal injections of 50 mg/kg DEN. The rats were sacrificed before start‐ ing DEN‐injections at 0 weeks, after 8 weeks, 14 weeks and 20 weeks after the start of DEN‐injec‐ tions. We performed histopathological, immunohistochemical, RT‐qPCR, RNA‐seq and flow cy‐ tometry analysis. Data were compared between tumor and non‐tumor samples from the DEN‐ treated versus untreated rats, as well as versus human HCCs. Chronic DEN injections lead to liver damage, hepatocytes proliferation, liver fibrosis and cirrhosis, disorganized vasculature, and a modulated immune microenvironment that mimics the usual events observed during human HCC development. The RNA‐seq results showed that DEN‐induced liver tumors in the rat model shared remarkable molecular characteristics with human HCC, especially with HCC associated with high proliferation. In conclusion, our study provides detailed insight into hepatocarcinogenesis in a com‐ monly used model of HCC, facilitating the future use of this model for preclinical testing. Citation: Kurma, K.; Manches, O.; Chuffart, F.; Sturm, N.; Gharzeddine, K.; Zhang, J.; Mercey‐ Ressejac, M.; Rousseaux, S.; Millet, A.; Lerat, H.; et al. DEN‐Induced Rat Model Reproduces Key Features of Human Hepatocellular Carcinoma. Cancers 2021, 13, 4981. https:// doi.org/10.3390/cancers13194981 Academic Editors: Vangelis Kondylis and Mathias Heikenwälder Received: 27 August 2021 Accepted: 30 September 2021 Published: 4 October 2021 Publisher’s Note: MDPI stays neu‐ tral with regard to jurisdictional claims in published maps and institu‐ tional affiliations. Copyright: © 2021 by the authors. Li‐ censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con‐ ditions of the Creative Commons At‐ tribution (CC BY) license (http://crea‐ tivecommons.org/licenses/by/4.0/).